Compositions and Methods for Managing Respiratory Conditions

ABSTRACT

This disclosure relates to compositions for use in managing respiratory distress and related disorders. In certain embodiments, the disclosure relates to methods of treating or preventing respiratory distress comprising administering an effective amount of a pharmaceutical composition comprising peptides or agents disclosed herein to a subject in need thereof. In certain embodiments, the peptides or agents decrease the concentration of claudin-5 in cells and tissues of the lungs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/485,552 filed Apr. 14, 2017. The entirety of this application ishereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under R01HL116958awarded by the National Institutes of Health. The government has certainrights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS A TEXT FILE VIA THEOFFICE ELECTRONIC FILING SYSTEM (EFS-WEB)

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 17072US ST25.txt. The text file is 8 KB, wascreated on Apr. 13, 2018, and is being submitted electronically viaEFS-Web.

BACKGROUND

The lung is the essential respiratory organ. The principal function isto transport oxygen from the atmosphere into the bloodstream and torelease carbon dioxide from the bloodstream into the atmosphere. Inorder for gas exchange to occur, the lung maintains a highly specializedbarrier between the atmosphere and fluid-filled tissues. The terminalairspaces of the lung (alveoli), where gas exchange occurs, provide thisbarrier. The alveoli are covered by a layer of epithelial cells, alveoliepithelial cells (AECs). AECs act as barrier to fluid leakage andregulate ion transport to promote absorption from the alveolar space toenable proper gas exchange. Conversely, insults that compromise thealveolar epithelial barrier promote accumulation of fluid within the airspace that severely compromises respiration.

Acute respiratory distress syndrome (ARDS) is characterized by a severedeficiency of oxygen in the bloodstream caused by alveolar inflammationresulting in the accumulation of fluid in the airspaces. ARDS isassociated with mortality. Thus, there is a need to identify therapeuticstrategies.

Wang et al. report heterogeneity of claudin expression by alveolarepithelial cells. Am. J. Respir. Cell. Mol. Biol. 29, 62-70 (2003).

Fernandez et al. report chronic alcohol ingestion alters claudinexpression in the alveolar epithelium of rats. Alcohol 41, 371-379(2007).

Baumgartner et al. report D-peptide analog of the second extracellularloop of claudin-3 and -4 leads to mislocalized claudin and cellularapoptosis in mammary epithelial cells. Chem. Biol. Drug Des. 77, 124-136(2011).

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to compositions for use in managing respiratorydistress and related disorders. In certain embodiments, the disclosurerelates to methods of treating or preventing respiratory distresscomprising administering an effective amount of a pharmaceuticalcomposition comprising peptides or agents disclosed herein to a subjectin need thereof. In certain embodiments, the peptides or agents decreasethe concentration of claudin-5 in cells and tissues of the lungs.

In certain embodiments, the peptides are claudin-5 mimetic peptides withan amino acid sequence corresponding to the region of the secondextracellular (E2) domain adjacent to the third transmembrane (TM3)domain of claudin-5. In certain embodiments, this disclosure relates toa peptide having SEQ ID NO: 1 (EFYDP), derivatives, prodrugs, or saltsthereof. In certain embodiments, proline (P) is a D-isomer, asparticacid (D) is a D-isomer, tyrosine (Y) is a D-isomer, phenylalanine (F) isa D-isomer, glutamic acid (E) is a D-isomer, or combinations thereof. Incertain embodiments, all of the amino acids are D-isomers.

In certain embodiments, the pharmaceutical agents are interfering RNAsuch short hairpin RNA (shRNA) that decrease claudin-5 expression byalveolar epithelial cells (AECs).

In certain embodiments, the disclosure contemplates the use of peptidesor agents that prevent claudin-5 from causing claudin-18 and otherbarrier forming claudins to dissociate from ZO-1 and other proteins thatpromote claudins to form barriers. In certain embodiments, thedisclosure contemplates the use of peptides or agents disclosed hereinthat prevent claudin-18 to dissociate from ZO-1. It is also contemplatedthat claudin-5 disrupts other claudins besides claudin-18.

In certain embodiments, this disclosure relates to methods of managingrespiratory conditions or diseases using pharmaceutical agents thatdecrease in the number of alveolar epithelial cells (AECs) withtight-junction spikes using agents such as Dynasore (chemical name,N′-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthohydrazide) or saltsthereof.

In certain embodiments, the disclosure contemplates compositionscomprising peptides or agents disclosed herein and a pharmaceuticallyacceptable excipient. In certain embodiments, the disclosurecontemplates pharmaceutical composition comprising a peptide, or otherpharmaceutical agent disclosed herein, or pharmaceutically acceptablesalts thereof, and a pharmaceutically acceptable excipient. In certainembodiments, the pharmaceutical composition is in the form of asterilized pH buffered aqueous salt solution or a saline phosphatebuffer between a pH of 6 to 8, optionally comprising a saccharide orpolysaccharide. In certain embodiments, the pharmaceutically acceptableexcipient are aerosolizing agents, phospholipids or pulmonarysurfactants. In certain embodiments, the aerosolizing agent is ahydrofluoroalkane, 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoropropane, propane, n-butane, isobutene, carbondioxide, air, nitrogen, nitrous oxide, dimethyl ether,trans-1,3,3,3-tetrafluoroprop-1-ene, or combinations thereof. In certainembodiments, the phospholipids are dipalmitoylphosphatidylcholine,palmitoyl-oleoyl phosphatidylglycerol, phosphatidylglycerol, orcombinations thereof.

In certain embodiments, the disclosure contemplates methods of treatingor preventing respiratory distress comprising administering an effectiveamount of a pharmaceutical composition comprising a peptide or agentdisclosed herein to a subject in need thereof. In certain embodiments,the subject is diagnosed with alcoholic lung syndrome; acute respiratorydistress syndrome; sepsis-associated lung disorders; bacterial and viralpneumonia; ventilator induced lung injury; bronchopulmonary dysplasia(BPD); asthma; bronchial, allergic, intrinsic, extrinsic or dust asthma;chronic or inveterate asthma; late asthma or airwayshyper-responsiveness; chronic obstructive pulmonary disease (COPD);allergic rhinitis; bronchitis; emphysema; or cystic fibrosis. In certainembodiments, the peptide is administered in combination with anotherrespiratory agent, anti-inflammatory agent, or antibiotic.

In certain embodiments, the disclosure contemplates recombinant nucleicacids vectors and cells comprising a nucleic acid that encodes peptidesdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows immunoblot data indicating alcohol exposure significantlydecreased claudin-4 expression (n=3, *P=0.002, t-test) and significantlyincreased claudin-5 expression (n=3, #P=0.005, t-test) by alveolarepithelial cells (AECs).

FIG. 1B shows data indicating yellow fluorescent protein (YFP)-claudin-5at MOI of 5 significantly increased claudin-5 expression (n=3, *P=0.022,one way ANOVA with Tukey multiple comparisons test). Control AECs weretransduced with adenovector YFP-claudin-5 at MOI of 2.5 or 5 or withEGFP adenovector at MOI of 5 as a control.

FIG. 1C shows data indicating decreased transepithelial resistance(TER). TER of alcohol exposed cells was significantly lower thancomparable control cells (n=3, #P=0.0005 versus EGFP transduced controlAECs; †P=0.028 versus EGFP transduced alcohol exposed cells, one wayANOVA, n=3, *P=0.036 control vs alcohol, one way ANOVA).

FIG. 1D shows data indicating Claudin-5 was significantly depleted byspecific shRNAs versus scrambled shRNA treated cells (n=4, *P=0.006,#P=0.036, one way ANOVA). Claudin-5 protein expression inalcohol-exposed AECs was depleted using a lentiviral system deliveringshRNA targeting claudin-5 or control scrambled shRNAs.

FIG. 1E shows data indicating decreased claudin-5 expression inalcohol-exposed cells significantly increased TER as compared with cellstransduced with scrambled shRNAs (n=4, #P<0.001, †P<0.001, one wayANOVA). TER of cells from alcohol exposed cells treated with shRNA wassignificantly lower than comparable control cells (n=4, *P<0.001, oneway ANOVA).

FIG. 2A shows control versus alcohol data on the quantification of the %of cells containing 3 or more tight junction spikes oriented towards thenucleus demonstrated that alcohol exposed and YFP-claudin-5 transducedAECs had significantly more spikes than comparable controls (*P=0.035,unpaired two-tailed t-test, n=11 fields).

FIG. 2B shows data on EGFP versus YFP-claudin-5 (*P<0.001, unpairedtwo-tailed t-test, n=11 fields).

FIG. 2C shows data indicating alcohol exposed AECs transduced withclaudin-5 shRNA1 had significantly fewer spikes than cells treated withcontrol shRNA (*P=0.011, one way ANOVA with Tukey multiple comparisonstest, n=5 fields). Cells treated with shRNA2 showed a trend towardsdecreased spikes (#P=0.18, one way ANOVA with Tukey multiple comparisonstest, n=5).

FIG. 3A shows data on the quantification of the % cells containing 3 ormore tight junction spikes oriented towards the nucleus demonstratedthat 160 μM Dynasore significantly decreased the number of cells fromalcohol fed rats containing spikes (n=8-9 fields, *P=0.002, one wayANOVA with Tukey multiple comparisons test).

FIG. 3B shows data indicating Dynasore did not have a significant effecton spike formation by control cells n=8-9 fields, *P=0.002, one wayANOVA with Tukey multiple comparisons test).

FIG. 4A shows data on the quantification of co-localization usingstochastic optical reconstruction microscopy (STORM) images demonstrateda significant change. In alcohol-exposed AECs there was a significantdecrease in ZO-1:claudin-18 (n=4 fields (control), n=3 fields (alcoholexposed AECs) *P=0.014, unpaired two-tailed t-test).

FIG. 4B shows data indicating a correlation with a significant increasein claudin-18:claudin-5 co-localization using STORM images (n=3 fields,*P=0.039, unpaired two-tailed t-test).

FIG. 4C shows data indicating ZO-1:claudin-5 co-localization wasunchanged (n=4 fields, unpaired two-tailed t-test).

FIG. 5A shows data on the quantification of co-localization using PLAdemonstrated a significant change. In alcohol-exposed AECs there was asignificant decrease in ZO-1:claudin-18 (n=6 fields,*P=0.018, unpairedtwo-tailed t-test).

FIG. 5B shows data indicating a correlation with a significant increasein claudin-18:claudin-5 co-localization (n=10 fields, *P=0.026, unpairedtwo-tailed t-test)

FIG. 5C show data indicating ZO-1:claudin-5 co-localization wasunchanged (n=6 fields, unpaired two-tailed t-test).

FIG. 6A shows data of biochemical analysis of protein insolubility wasassessed by a Triton X-100 solubilization assay comparing control AECsto YFP-claudin-5 transduced cells. At 6 days in culture, AECs wereharvested and extracted using 0.1% Triton X-100, an aliquot of totalprotein (T) was set aside and the remainder was centrifuged to separateTriton X-100 soluble (S) and insoluble (I) fractions that were measuredby immunoblot for claudin-18. Quantification of the soluble fractionrevealed that YFP-claudin-5 expression significantly increasedclaudin-18 solubility from 35.2%±1.8 to 42.1%±0.6 while claudin-5 andZO-1 solubility did not significantly change (n=3, *P=0.003, unpairedtwo-tailed t-test).

FIG. 6B shows data for claudin-5 (n=3, *P=0.003, unpaired two-tailedt-test).

FIG. 6C shows data for ZO-1 (n=3, *P=0.003, unpaired two-tailed t-test).

FIG. 7A shows data on TER for regular AECs in the presence of controlAc-LYQY(SEQ ID NO:35)-NH₂ and in the presence of Ac-EFYDP (SEQ IDNO:1)-NH₂ (C5). AECs isolated from control were cultured on Transwellpermeable supports for 5 days and then either untreated (un), orincubated with 10 mM control peptide (con; Ac-LYQY(SEQ ID NO:35)-NH₂) ora claudin-5 extracellular domain mimetic peptide (C5; Ac-EFYDP (SEQ IDNO:1)-NH₂) for 16 h. The cells were examined for barrier function bytransepithelial resistance (TER).

FIG. 7B shows data on TER for alcohol-exposed AECs in the presence ofcontrol Ac-LYQY(SEQ ID NO:35)-NH₂ and in the presence of Ac-EFYDP (SEQID NO:1)-NH₂ (C5) indicating that this claudin-5 extracellular domainmimetic increases barrier function of alcohol-exposed AECs (*P=0.014versus untreated; #P=0.042 versus control; n=6, one way ANOVA with Tukeymultiple comparisons test).

FIG. 7C shows data on paracellular flux of calcein for regular AECs inthe presence of control Ac-LYQY(SEQ ID NO:35)-NH₂ and in the presence ofAc-EFYDP (SEQ ID NO:1)-NH₂ (C5).

FIG. 7D shows data on paracellular flux of calcein for alcohol-exposedAECs in the presence of control Ac-LYQY(SEQ ID NO:35)-NH₂ and in thepresence of Ac-EFYDP (SEQ ID NO:1)-NH₂ (C5) (*P=0.007 versus untreated;#P=0.054 versus control; n=3, one way ANOVA with Tukey multiplecomparisons test).

FIG. 7E shows data on paracellular flux of 10 kDa Texas Red dextran forregular AECs in the presence of control Ac-LYQY(SEQ ID NO:35)-NH₂ and inthe presence of Ac-EFYDP (SEQ ID NO:1)-NH₂ (C5).

FIG. 7F shows data on paracellular flux of 10 kDa Texas Red dextran foralcohol-exposed AECs in the presence of control Ac-LYQY(SEQ IDNO:35)-NH₂ and in the presence of Ac-EFYDP (SEQ ID NO:1)-NH₂ (C5)(*P=0.009 versus untreated; #P=0.040 versus control; n=3, one way ANOVAwith Tukey multiple comparisons test).

FIG. 7G shows data on cells from alcohol fed rats showed a decrease intight junction spikes that was significantly less than that of untreatedcontrols and alcoholic AECs that were either untreated or treated with acontrol peptide (*P<0.001 versus untreated; #P<0.001 versus controlpeptide; †P=0.041 versus untreated control AECs, n=9-11 fields from twoindependent experiments, one way ANOVA with Tukey multiple comparisonstest).

FIG. 7H shows data for control AECs were processed and examined byimmunoblot for claudin-5, claudin-18 and ZO-1.

FIG. 7I shows data for alcohol-exposed AECs as treated above wereprocessed and examined by immunoblot for claudin-5, claudin-18 and ZO-1.Cells from alcohol-fed rats that were treated with the C5 peptide showeda significant and specific decrease in claudin-5 (*P=0.042 versusuntreated; #P=0.016 versus control; n=9, one way ANOVA with Tukeymultiple comparisons test).

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise.

“Subject” refers any animal, preferably a human patient, livestock, ordomestic pet.

The terms “protein” and “peptide” refer to compounds comprising aminoacids joined via peptide bonds and are used interchangeably. Amino acidsmay be naturally or non-naturally occurring. A “chimeric protein” or“fusion protein” is a molecule in which different portions of theprotein are derived from different origins such that the entire moleculeis not naturally occurring. A chimeric protein may contain amino acidsequences from the same species of different species as long as they arenot arranged together in the same way that they exist in a naturalstate. Examples of a chimeric protein include sequences disclosed hereinthat are contain one, two or more amino acids attached to the C-terminalor N-terminal end that are not identical to any naturally occurringprotein, such as in the case of adding an amino acid containing an amineside chain group, e.g., lysine, an amino acid containing a carboxylicacid side chain group such as aspartic acid or glutamic acid, apolyhistidine tag, e.g. typically four or more histidine amino acids.Contemplated chimeric proteins include those with self-cleaving peptidessuch as P2A-GSG. See Wang. Scientific Reports 5, Article number: 16273(2015).

As used herein, the term “derivative” refers to a structurally similarpeptide that retains sufficient functional attributes of the identifiedanalogue. The derivative may be structurally similar because it islacking one or more atoms, e.g., replacing an amino group, hydroxyl, orthiol group with a hydrogen, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, replacing a oxygenatom with a sulfur atom or replacing an amino group with a hydroxylgroup. The derivative may be a prodrug, comprise a lipid, polyethyleneglycol, saccharide, polysaccharide. A derivative may be two or morepeptides linked together by a linking group. It is contemplated that thelinking group may be biodegradable. Derivatives may be prepared by anyvariety of synthetic methods or appropriate adaptations presented insynthetic or organic chemistry text books, such as those provide inMarch's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Wiley, 6th Edition (2007) Michael B. Smith or DominoReactions in Organic Synthesis, Wiley (2006) Lutz F. Tietze herebyincorporated by reference.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“═O”), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb,—NRaC(═O)NRaNRb, —NRaC(═O)ORb, —NRaSO₂Rb, —C(═O)Ra, —C(═O)ORa,—C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)₂Ra, —OS(═O)₂Ra and—S(═O)₂ORa. Ra and Rb in this context may be the same or different andindependently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino,alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,and heteroarylalkyl. The substituents may further optionally besubstituted.

As used herein, a “lipid” group refers to a hydrophobic group that isnaturally or non-naturally occurring that is highly insoluble in water.As used herein a lipid group is considered highly insoluble in waterwhen the point of connection on the lipid is replaced with a hydrogenand the resulting compound has a solubility of less than 0.63×10⁻⁴% w/w(at 25° C.) in water, which is the percent solubility of octane in waterby weight. See Solvent Recovery Handbook, 2^(nd). Ed, Smallwood, 2002 byBlackwell Science, page 195. Examples of naturally occurring lipidsinclude saturated or unsaturated hydrocarbon chains found in fattyacids, glycerolipids, cholesterol, steroids, polyketides, andderivatives. Non-naturally occurring lipids include derivatives ofnaturally occurring lipids, acrylic polymers, aromatic, and alkylatedcompounds and derivatives thereof.

The term “prodrug” refers to an agent that is converted into abiologically active form in vivo. Prodrugs are often useful because, insome situations, they may be easier to administer than the parentcompound. The prodrug may also have improved solubility inpharmaceutical compositions over the parent drug. A prodrug may beconverted into the parent drug by various mechanisms, includingenzymatic processes and metabolic hydrolysis. Typical prodrugs arepharmaceutically acceptable esters. Prodrugs include compounds whereinan hydroxy, amino or mercapto (thiol) group is bonded to any group that,when the prodrug of the active compound is administered to a subject,cleaves to form a free hydroxy, free amino or free mercapto group,respectively. Examples of prodrugs include, but are not limited to,acetate, formate and benzoate derivatives of an alcohol or acetamide,formamide and benzamide derivatives of an amine functional group in theactive compound and the like.

For example, if a disclosed peptide or a pharmaceutically acceptableform of the peptide contains a carboxylic acid functional group, aprodrug can comprise a pharmaceutically acceptable ester formed by thereplacement of the hydrogen atom of the acid group with a group such as(C₁-C₈)alkyl, (C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl havingfrom 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbonatoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as beta-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

If a disclosed peptide or a pharmaceutically acceptable form of thepeptide contains an alcohol functional group, a prodrug can be formed bythe replacement of the hydrogen atom of the alcohol group with a groupsuch as (C₁-C₆)alkanoyloxymethyl, 1-((C₁-C₆)alkanoyloxy) ethyl,1-methyl-1((C₁-C₆)alkanoyloxy)ethyl (C₁-C₆)alkoxycarbonyloxymethyl,—N—(C₁-C₆)alkoxycarbonylaminomethyl, succinoyl, (C₁-C₆)alkanoyl,alpha-amino(C₁-C₄)alkanoyl, arylacyl and alpha-aminoacyl, oralpha-aminoacyl-alpha-aminoacyl, where each alpha-aminoacyl group isindependently selected from amino acids P(O)(OH)₂,—P(O)(O(C₁-C₆)alkyl)₂, and glycosyl (the radical resulting from theremoval of a hydroxyl group of the hemiacetal form of a carbohydrate).

If a disclosed peptide or a pharmaceutically acceptable form of thepeptide incorporates an amine functional group, a prodrug can be formedby the replacement of a hydrogen atom in the amine group with a groupsuch as R-carbonyl, RO-carbonyl, NRR′-carbonyl where R and R′ are eachindependently (C₁-C₁₀)alkyl, (C₃-C₇)cycloalkyl, benzyl, a naturalalpha-aminoacyl, —C(OH)C(O)OY₁ wherein Y¹ is H, (C₁-C₆)alkyl or benzyl,—C(OY₂)Y₃ wherein Y₂ is (C₁-C₄) alkyl and Y₃ is (C₁-C₆)alkyl,carboxy(C₁-C₆)alkyl, amino(C₁-C₄)alkyl or mono-Nordi-N,N—(C₁-C₆)alkylaminoalkyl, —C(Y₄)Y₅ wherein Y₄ is H or methyl and Y₅is mono-N- or di-N,N—(C₁-C₆)alkylamino, morpholino, piperidin-1-yl orpyrrolidin-1-yl.

As used herein, “pharmaceutically acceptable esters” include, but arenot limited to, alkyl, alkenyl, alkynyl, aryl, arylalkyl, and cycloalkylesters of acidic groups, including, but not limited to, carboxylicacids, phosphoric acids, phosphinic acids, sulfonic acids, sulfinicacids, and boronic acids.

As used herein, “pharmaceutically acceptable enol ethers” include, butare not limited to, derivatives of formula —C═C(OR) where R can beselected from alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl.Pharmaceutically acceptable enol esters include, but are not limited to,derivatives of formula —C═C(OC(O)R) where R can be selected fromhydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl.

A “linking group” refers to any variety of molecular arrangements thatcan be used to bridge to molecular moieties together. An example formulamay be —R_(m)— wherein R is selected individually and independently ateach occurrence as: —CR_(m)R_(m)—, —CHR_(m)—, —CH—, —C—, —CH₂—,—C(OH)R_(m), —C(OH)(OH)—, —C(OH)H, —C(Hal)R_(m)—, —C(Hal)(Hal)-,—C(Hal)H—, —C(N₃)R_(m)—, —C(CN)R_(m)—, —C(CN)(CN)—, —C(CN)H—,—C(N₃)(N₃)—, —C(N₃)H—, —O—, —S—, —N—, —NH—, —NR_(m)—, —(C═O)—, —(C═NH)—,—(C═S)—, —(C═CH₂)—, which may contain single, double, or triple bondsindividually and independently between the R groups. If an R is branchedwith an R_(m) it may be terminated with a group such as —CH₃, —H,—CH═CH₂, —CCH, —OH, —SH, —NH₂, —N₃, —CN, or -Hal, or two branched Rs mayform a cyclic structure. It is contemplated that in certain instances,the total Rs or “m” may be less than 100, or 50, or 25, or 10. Examplesof linking groups include bridging alkyl groups and alkoxyalkyl groups.Linking groups may be substituted with one or more substituents.

As used herein, the term “biodegradable” in reference to a substituentor linker refers to a molecular arrangement in a peptide derivative thatwhen administered to a subject, e.g., human, will be broken down bybiological mechanism such that a metabolite will be formed and themolecular arrangement will not persist for over a long period of time,e.g., the molecular arrangement will be broken down by the body after aseveral hours or days. In certain embodiments, the disclosurecontemplates that the biodegradable linker or substituent will not existafter a week or a month.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, the term “sterilized” refers to subjecting something toa process that effectively kills or eliminates transmissible agents(such as fungi, bacteria, viruses, prions and spore forms etc.).Sterilization can be achieved through application of heat, chemicals,irradiation, high pressure or filtration. One process involves waterprepared by distillation and stored in an airtight container whereinsuitable additives are introduced to approximate isotonicity.

The term “polynucleotide” refers to a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The polynucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. The term “oligonucleotide” generally refers to a short lengthof single-stranded polynucleotide chain usually less than 30 nucleotideslong, although it may also be used interchangeably with the term“polynucleotide.”

The term “nucleic acid” refers to a polymer of nucleotides, or apolynucleotide, as described above. The term is used to designate asingle molecule, or a collection of molecules. Nucleic acids may besingle stranded or double stranded, and may include coding regions andregions of various control elements.

A “heterologous” nucleic acid sequence or peptide sequence refers to anucleic acid sequence or peptide sequence that do not naturally occur,e.g., because the whole sequences contain a segment from other plants,bacteria, viruses, other organisms, or joinder of two sequences thatoccur the same organism but are joined together in a manner that doesnot naturally occur in the same organism or any natural state.

Regulation of Claudin/Zonula Occludens-1 Complexes by Hetero-ClaudinInteractions

Although there are a number of diseases and conditions that can lead toacute respiratory distress syndrome (ARDS), studies identified alcoholabuse emerged as the only independent risk factor known to increase theodds of any given at-risk individual developing ARDS. Chronic alcoholconsumption is associated with diminished lung barrier function inpatients.

Tight junctions in the lungs are composed of several different classesof transmembrane, cytosolic and cytoskeletal proteins which interact ina coordinated manner to form epithelial barriers at cell to cell contactregions. Claudins, a family of tetraspan transmembrane proteins with 27members, form the structural basis for control of tight junctionpermeability. They associate with other claudins in adjacent cells andseal the gaps between them to create a regulated barrier to paracellulardiffusion across the epithelia. Interactions between claudins areimportant for barrier function. In order to form a functional barrier,claudins interact with each other within the same cellular membrane(polymeric cis-interactions) and between two cells via head-to-headinteractions (polytypic trans-interactions). Claudin interactions can beclassified as homomeric (polymeric cis-interaction) and homotypic(trans-interaction) when the polymer consists of the same claudin.

Over a dozen different claudins have been found in the alveolarepithelium including claudin-1, -3, -4, -5, -7 and -18. Claudinstructure plays an important role in tight junction formation. Claudinshave four transmembrane domains, two extracellular loops, oneintracellular loop as well as a cytoplasmic localized N- and C-terminus.

Dietary alcohol significantly impairs alveolar epithelial cell (AEC)tight junctions that are required to provide a barrier betweenfluid-filled tissues and the airspace. However, the molecular basis forthe effects of alcohol on alveolar epithelial tight junctions is notwell understood. Isolated primary rat AECs that differentiate into amodel type-I monolayer were studied at a molecular level. Rats feddietary alcohol for 8 weeks provide an animal model system thatrecapitulates the pathologic consequences of chronic alcohol ingestionon lung barrier function. Moreover, primary cells derived fromalcohol-fed rats (alcohol-exposed AECs) have impaired barrier functionthat persists in vitro, as compared with ABCs isolated from animals fedan isocaloric control diet.

AECs from alcohol-fed animals have significant changes in tight-junctionprotein expression that are associated with a decrease in epithelialbarrier function. Among these changes is an increase in claudin-5expression. By molecular manipulation of AECs, it was discovered thatclaudin-5 is both necessary and sufficient to disrupt AEC tightjunctions. Increased claudin-5 expression induces the formation ofclaudin-containing structures perpendicular to the axis of the cell-cellinterface (tight-junction spikes) that are active sites of vesiclebudding and fusion. The appearance of tight-junction spikes correlateswith increased paracellular leak between AECs. Using severalcomplementary approaches, it was discovered that claudin-5 interactedwith claudin-18, and that this decreases the ability of claudin-18 toproductively interact with zonula occludens 1 (ZO-1). This mechanism istargetable using a claudin-5 mimetic peptide, indicating a potentialtherapeutic approach to promote alveolar barrier function.

Increased claudin-18:claudin-5 interactions decreased ZO-1:claudin-18co-localization, which correlated with weakened assembly into tightjunctions as evidenced by an increase in Triton X-100 solubility. Thenet effect of decreased interactions between claudin-18 and ZO-1 is todestabilize tight junctions that, in turn, increases paracellular leak.

Two examples of claudin-claudin interactions that occur before deliveryto the plasma membrane are claudin-4:claudin-8, andclaudin-16:claudin-19. In each of those cases, depletion or misfoldingof one claudin resulted in intracellular accumulation of the other,evidence that these pairs of claudins serve as co-chaperones.Interestingly, in kidney epithelia, claudin-18 trafficking wasindependent of claudin-16 and claudin-19, indicating specificity ofcis-claudin interactions. In AECs, the intracellular pools of claudin-5and claudin-18 are limited, largely vesicular and do not show completeco-localization. As the effects of claudin-5 on claudin-18 largelyaffect tight-junction morphology in AECs, and that these effects areantagonized by a claudin-5 extracellular mimetic peptide, it seems morelikely to be that claudin-5 and claudin-18 interact within tightjunctions or other regions of the plasma membrane rather than beforedelivery. Considering that tight-junction-associated claudins are highlydynamic there is certainly the capacity for claudin remodelling to occurwithin pre-formed tight junctions at cell-cell interfaces as well as inclaudins newly delivered to the plasma membrane.

Cis interactions between claudin-5 and claudin-18 can diminish barrierfunction by affecting the ability of claudin-18 to form complexes withZO-1. Cis interactions between claudin-5 and claudin-3 have previouslybeen characterized at a molecular level. It was unknown that claudin-5can regulate the ability of claudin-18, to interact with the cytoplasmicscaffold including claudin-5 interfering with other lung claudins.

Claudin-5 increased formation of tight-junction spikes that, in turn,correlated with increased paracellular leak. Association oftight-junction spikes with increased paracellular permeability isconsistent with reports demonstrating that spikes and barrierdysfunction are also induced by transforming growth factor-β1 andnuclear factor-κB inhibitors. Normal AECs treated with the nuclearfactor-κB inhibitor BMS-345541 showed both increased claudin-5expression and increased formation of tight-junction spikes as a resultof interfering with granulocyte-macrophage colony-stimulating factorsignaling that mimics the effects of alcohol on AECs. Here, live-cellimaging was used to confirm that these were sites whereclaudin-containing vesicles were observed to bud and fuse from the endsof spikes. Linking tight-junction spikes and enhanced endocytosis with adecrease in barrier function is also consistent with treatment of fetalAECs with endocytosis inhibitors almost doubled transepithelialresistance (TER).

Given that tight-junction spikes are associated with alcohol andclaudin-5 expression, and that these are sites of active vesicletrafficking of claudin-containing vesicles, increased claudin-5 accountsfor the deleterious effects of dietary alcohol on AEC barrier function.The effects of increased claudin-5 appear to contradict the role ofclaudin-5 in promoting endothelial barrier function. Data hereinindicates that claudin-5 function is cell type dependent and isinfluenced by the context of expression. For example, claudin-5 has thecapacity to increase barrier function of MDCK II cells, which areotherwise exceptionally leaky, with baseline TER in the range of 100Ω×cm2. In AECs, which are much tighter, claudin-5 had the opposite effect.It is also possible that the ability of claudin-5 to impair tightjunctions is specifically dependent on an interaction with claudin-18,which is not present in MDCK cells. A specific interaction betweenclaudin-5 and claudin-18 has particular relevance to alveolar barrierfunction. Although it is not intended that embodiments of thisdisclosure be limited by any particular mechanism, the mechanism bywhich alcohol induces claudin-5 expression is under investigation atpresent and could either be transcriptional or posttranslational.

As claudin-5 has a dramatic effect on AEC barrier function, itrepresents an appealing potential pharmacologic target to improvealveolar barrier function in vulnerable individuals, using a claudin-5mimetic peptide (Ac-EFYDP (SEQ ID NO: 1)-NH₂). The feasibility of thisapproach was confirmed. The peptide specifically increased barrierfunction of alcohol-exposed AECs. The D-amino acid version of anAc-DFYNP-NH₂ mimetic is 10-100-fold more effective than thecorresponding L-amino acid version. Unlike the DFYNP sequence that isshared by several claudins important for lung barrier function,including claudin-3 and claudin-4, the EFYDP (SEQ ID NO: 1)corresponding to claudin-5 is unlikely to cross-react with othernon-homologous claudins and claudin-1 expression in the lung is low andunaffected by the peptide.

EFYDP (SEQ ID NO: 1) is in the E2 region of the protein directlyadjacent to the TM3 domain, a region of claudin-5 that mediatescis-claudin interactions which is consistent with the idea thatclaudin-5 interactions with claudin-18 have a deleterious effect on theability of claudin-18 to interact with ZO-1. The ability of acis-claudin interaction to affect interactions of another claudin withthe tight-junction scaffold represents a novel mode of tight-junctionregulation with the potential to be pharmacologically manipulable.Specific and direct targeting of claudin-5 using these approaches offersthe potential of preventing acute respiratory distress syndrome,particularly in those individuals at greatest risk due to underlyingalcohol abuse, by improving alveolar barrier function and fluidclearance.

Peptide Mimetics, Derivatives, and Prodrugs

In certain embodiments, the pharmaceutical agents are claudin-5 mimeticpeptides corresponding to the region of the second extracellular (E2)domain adjacent to the third transmembrane (TM3) domain of claudin-5. Incertain embodiments, this disclosure relates to methods ofpharmaceutical management using pharmaceutical agents such as a peptidehaving SEQ ID NO: 1 (EFYDP), derivatives, prodrugs, or salts thereof. Incertain embodiments, proline (P) is a D-isomer, aspartic acid (D) is aD-isomer, tyrosine (Y) is a D-isomer, phenylalanine (F) is a D-isomer,glutamic acid (E) is a D-isomer, or combinations thereof. In certainembodiments, all of the amino acids are D-isomers.

In certain embodiments, a peptide derivative is one where glutamic acid(E) comprises an N-terminal alkanoyl group optionally substituted withone or more substituents. In certain embodiments, a peptide derivativeis one where glutamic acid (E) comprises an alkyl carboxy ester groupoptionally substituted with one or more substituents. In certainembodiments, a peptide derivative is one where phenylalanine (F)comprises a phenyl group optionally substituted with one or moresubstituents. In certain embodiments, a peptide derivative is one whereaspartic acid (D) comprises an alkyl carboxy ester group optionallysubstituted with one or more substituents. In certain embodiments, apeptide derivative is one where proline (P) comprises a C-terminal amideoptionally substituted with one or more substituents.

In certain embodiments, the disclosure contemplates peptides disclosedherein having at least one molecular modification, e.g., such that thepeptide contains a non-naturally amino acid. In certain embodiments, thedisclosure contemplates a non-naturally occurring derivative of apeptide having SEQ ID NO: 1. In certain embodiments, the disclosurecontemplates a derivative in the form of a prodrug. In certainembodiments, the disclosure contemplates a derivative wherein an amino,carboxyl, or hydroxyl group in a peptide disclosed herein issubstituted. In certain embodiments, the disclosure contemplatespeptides disclosed herein having a label, e.g., fluorescent orradioactive. In certain embodiments, the peptide comprises anon-naturally occurring amino acid. In certain embodiments, the peptidecomprises an amino acid comprising a halogen.

In certain embodiments, the disclosure contemplates derivatives of SEQID NO: 1 (EFYDP) that are amino acid substitutions wherein amino acid Ecan be D; F can be Y or W; and Y can be F or W.

In certain embodiments, a peptide of this disclosure has the followingformula

derivatives, prodrugs, esters, or salts thereof.

The peptides of this disclosure may be made from using commerciallyavailable BOC and Fmoc protected amino acids using well-known syntheticprocedures for amide coupling, e.g., using solid phase resins. Acidhalides of amide coupling reactions can be used to N-terminally modifythe peptide. Carboxylic acids may be exposed to alcohols in the presenceof an acid to form esters. Carboxylic acids may be converted toactivated intermediates that react with ammonia or other substitutedamines to form C-terminal amides or amides of glutamic or aspartic acid.Alternatively, the peptides can be prepared by expression systems thatutilize recombinant nucleic acids encoding the peptides or fusionpeptides. The fusion peptides may be exposed to cleaving agents or thefusion peptides may contain self-cleaving sequences allowing the peptideproduct to be isolated and optionally further modified.

In certain embodiments, the peptides discloses herein have at least onenon-naturally occurring molecular modification, such as the attachmentof polyethylene glycol, the attachment of a chimeric peptide, theattachment of a fluorescent dye comprising aromatic groups, fluorescentpeptide, a chelating agent capable of binding a radionuclide such as¹⁸F, N-terminal acetyl, propionyl group, myristoyl and palmitoyl, groupor N-terminal methylation, or a C-terminal alkyl ester. In certainembodiments, the disclosure contemplates the disclosure contemplatespeptides disclosed herein labeled using commercially availablebiotinylation reagents. Biotinylated peptide can be used in streptavidinaffinity binding, purification, and detection.

In certain embodiments, this disclosure contemplates derivatives ofpeptide disclose herein wherein one or more amino acids are substitutedwith chemical groups to improve pharmacokinetic properties such assolubility and serum half-life, optionally connected through a linker.In certain embodiments, such a derivative may be a prodrug wherein thesubstituent or linker is biodegradable, or the substituent or linker isnot biodegradable. In certain embodiments, contemplated substituentsinclude a saccharide, polysaccharide, acetyl, fatty acid, lipid, and/orpolyethylene glycol. The substituent may be covalently bonded throughthe formation of amide bonds on the C-terminus or N-terminus of thepeptide optionally connected through a linker. In certain embodiments,it is contemplated that the substituent may be covalently bonded throughan amino acid within the peptide, e.g. through an amino acid containinga carboxylic acid side chain group such as aspartic acid or glutamicacid, within the peptide comprising a sequence disclosed herein.

In certain embodiments, the disclosure relates to recombinantpolypeptides comprising sequences disclosed herein or fusions thereofwherein the amino terminal end or the carbon terminal end of the aminoacid sequence are optionally attached to a heterologous amino acidsequence, label, or reporter molecule.

Thus, in certain embodiments, the disclosure contemplates recombinantnucleic acids vectors and cells comprising the same. In certainembodiments, the disclosure relates to expression systems comprising arecombinant vector comprising a nucleic acid encoding peptide disclosedherein. In certain embodiments, the disclosure relates to cellscomprising a recombinant vector comprising a nucleic acid encodingpeptide disclosed herein. In certain embodiments, the disclosure relatesto a vector comprising the nucleic acid encoding a peptide disclosedherein and a heterologous nucleic acid sequence.

The terms “vector” or “expression vector” refer to a recombinant nucleicacid containing a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism or expression system, e.g.,cellular or cell-free. Nucleic acid sequences necessary for expressionin prokaryotes usually include a promoter, an operator (optional), and aribosome binding site, often along with other sequences. Eukaryoticcells are known to utilize promoters, enhancers, and termination andpolyadenylation signals.

Protein “expression systems” refer to in vivo and in vitro (cell free)systems. Systems for recombinant protein expression typically utilizecells transfecting with a DNA expression vector that contains thetemplate. The cells are cultured under conditions such that theytranslate the desired protein. Expressed proteins are extracted forsubsequent purification. In vivo protein expression systems usingprokaryotic and eukaryotic cells are well known. Proteins may berecovered using denaturants and protein-refolding procedures. In vitro(cell-free) protein expression systems typically usetranslation-compatible extracts of whole cells or compositions thatcontain components sufficient for transcription, translation andoptionally post-translational modifications such as RNA polymerase,regulatory protein factors, transcription factors, ribosomes, tRNAco-factors, amino acids and nucleotides. In the presence of anexpression vectors, these extracts and components can synthesizeproteins of interest. Cell-free systems typically do not containproteases and enable labeling of the protein with modified amino acids.Some cell free systems incorporated encoded components for translationinto the expression vector. See, e.g., Shimizu et al., Cell-freetranslation reconstituted with purified components, 2001, Nat.Biotechnol., 19, 751-755 and Asahara & Chong, Nucleic Acids Research,2010, 38(13): e141, both hereby incorporated by reference in theirentirety.

A “selectable marker” is a nucleic acid introduced into a recombinantvector that encodes a polypeptide that confers a trait suitable forartificial selection or identification (report gene), e.g.,beta-lactamase confers antibiotic resistance, which allows an organismexpressing beta-lactamase to survive in the presence antibiotic in agrowth medium. Another example is thymidine kinase, which makes the hostsensitive to ganciclovir selection. It may be a screenable marker thatallows one to distinguish between wanted and unwanted cells based on thepresence or absence of an expected color. For example, the lac-z-geneproduces a beta-galactosidase enzyme that confers a blue color in thepresence of X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside). Ifrecombinant insertion inactivates the lac-z-gene, then the resultingcolonies are colorless. There may be one or more selectable markers,e.g., an enzyme that can complement to the inability of an expressionorganism to synthesize a particular compound required for its growth(auxotrophic) and one able to convert a compound to another that istoxic for growth. URA3, an orotidine-5′ phosphate decarboxylase, isnecessary for uracil biosynthesis and can complement ura3 mutants thatare auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid intothe toxic compound 5-fluorouracil. Additional contemplated selectablemarkers include any genes that impart antibacterial resistance orexpress a fluorescent protein. Examples include, but are not limited to,the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr,neomycin phosphotransferase type II gene (nptII), p-glucuronidase (gus),green fluorescent protein (gfp), egfp, yfp, mCherry, p-galactosidase(lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat),alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphosresistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase(xylA), arabitol dehydrogenase (at1D), UDP-glucose:galactose-1-phosphateuridyltransferasel (galT), feedback-insensitive α subunit ofanthranilate synthase (OASA1D), 2-deoxyglucose (2-DOGR),benzyladenine-N-3-glucuronide, E. coli threonine deaminase, glutamate1-semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO),salt-tolerance gene (rstB), ferredoxin-like protein (pflp),trehalose-6-P synthase gene (AtTPS1), lysine racemase (lyr),dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1(AtTSB1), dehalogenase (dhlA), mannose-6-phosphate reductase gene(M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase(dsdA).

A “label” refers to a detectable compound or composition that isconjugated directly or indirectly to another molecule, such as anantibody or a protein, to facilitate detection of that molecule.Specific, non-limiting examples of labels include fluorescent tags,enzymatic linkages, and radioactive isotopes. In one example, a “labelreceptor” refers to incorporation of a heterologous polypeptide in thereceptor. A label includes the incorporation of a radiolabeled aminoacid or the covalent attachment of biotinyl moieties to a polypeptidethat can be detected by marked avidin (for example, streptavidincontaining a fluorescent marker or enzymatic activity that can bedetected by optical or colorimetric methods). Various methods oflabeling polypeptides and glycoproteins are known in the art and may beused. Examples of labels for polypeptides include, but are not limitedto, the following: radioisotopes or radionucleotides (such as 35S or131I) fluorescent labels (such as fluorescein isothiocyanate (FITC),rhodamine, lanthanide phosphors), enzymatic labels (such as horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase),chemiluminescent markers, biotinyl groups, predetermined polypeptideepitopes recognized by a secondary reporter (such as a leucine zipperpair sequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags), or magnetic agents, such as gadolinium chelates.In some embodiments, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance.

In certain embodiments, the disclosure relates to the recombinantvectors comprising a nucleic acid encoding a polypeptide disclosedherein or fusion protein thereof. In certain embodiments, therecombinant vector optionally comprises a mammalian, human, insect,viral, bacterial, bacterial plasmid, yeast associated origin ofreplication or gene such as a gene or retroviral gene or lentiviral LTR,TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selectedfrom tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selectedfrom gag, pol, and env.

In certain embodiments, the recombinant vector optionally comprises agene vector element (nucleic acid) such as a selectable marker region,lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG)promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerasepromoter, SV40 promoter, internal ribosome entry site (IRES) sequence,cis-acting woodchuck post regulatory regulatory element (WPRE),scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAGtag coding region, c-myc tag coding region, metal affinity tag codingregion, streptavidin binding peptide tag coding region, polyHis tagcoding region, HA tag coding region, MBP tag coding region, GST tagcoding region, polyadenylation coding region, SV40 polyadenylationsignal, SV40 origin of replication, Col E1 origin of replication, f1origin, pBR322 origin, or pUC origin, TEV protease recognition site,loxP site, Cre recombinase coding region, or a multiple cloning sitesuch as having 5, 6, or 7 or more restriction sites within a continuoussegment of less than 50 or 60 nucleotides or having 3 or 4 or morerestriction sites with a continuous segment of less than 20 or 30nucleotides.

In certain embodiments, the disclosure relates to a nucleic acidencoding a polypeptide disclosed herein wherein the nucleotide sequencehas been changed to contain at least one non-naturally occurringsubstitution and/or modification relative to the naturally occurringsequence, e.g., one or more nucleotides have been changed relative tothe natural sequence. In certain embodiments, the disclosure relates toa nucleic acid encoding a polypeptide disclosed herein furthercomprising a label.

Interfering Claudin-5 mRNA Nucleobase Polymers

In certain embodiments, the disclosure contemplates inhibition ofclaudin-5 mRNA, e.g., the use of nucleobase polymers for antisensedisruptions or RNA interference of Claudin-5 mRNA expression orClaudin-5 mRNA binding in order to decrease Claudin-5 mRNA expression asa therapeutic strategy.

In certain embodiments, the disclosure contemplates methods of treatingor preventing respiratory distress comprising administering an effectiveamount of a pharmaceutical composition comprising a nucleobase polymersfor antisense disruptions or RNA interference of Claudin-5 mRNAexpression disclosed herein to a subject in need thereof. In certainembodiments, the subject is diagnosed with alcoholic lung syndrome;acute respiratory distress syndrome; sepsis-associated lung disorders;bacterial and viral pneumonia; ventilator induced lung injury;bronchopulmonary dysplasia (BPD); asthma; bronchial, allergic,intrinsic, extrinsic or dust asthma; chronic or inveterate asthma; lateasthma or airways hyper-responsiveness; chronic obstructive pulmonarydisease (COPD); allergic rhinitis bronchitis, emphysema; or cysticfibrosis. In certain embodiments, the peptide is administered incombination with another respiratory agent, anti-inflammatory agent, orantibiotic.

There are two human Claudin-5 mRNAs with NCBI accession numbersNM_001130861.1 and NM_003277.3. See Comely et al. Two common human CLDN5alleles encode different open reading frames but produce on proteinisoform. In NM_001130861, bases 1083-1739 which encode claudin-5 are(SEQ ID NO: 2) ATGGGGTCCGCAGCGTTGGAGATCCTGGGCCTGGTGCTGTGCCTGGTGGGCTGGGGGGGTCTGATCCTGGCGTGCGGGCTGCCCATGTGGCAGGTGACCGCCTTCCTGGACCACAACATCGTGACGGCGCAGACCACCTGGAAGGGGCTGTGGATGTCGTGCGTGGTGCAGAGCACCGGGCACATGCAGTGCAAAGTGTACGACTCGGTGCTGGCTCTGAGCACCGAGGTGCAGGCGGCGCGGGCGCTCACCGTGAGCGCCGTGCTGCTGGCGTTCGTTGCGCTCTTCGTGACCCTGGCGGGCGCGCAGTGCACCACCTGCGTGGCCCCGGGCCCGGCCAAGGCGCGTGTGGCCCTCACGGGAGGCGTGCTCTACCTGTTTTGCGGGCTGCTGGCGCTCGTGCCACTCTGCTGGTTCGCCAACATTGTCGTCCGCGAGTTTTACGACCCGTCTGTGCCCGTGTCGCAGAAGTACGAGCTGGGCGCAGCGCTGTACATCGGCTGGGCGGCCACCGCGCTGCTCATGGTAGGCGGCTGCCTCTTGTGCTGCGGCGCCTGGGTCTGCACCGGCCGTCCCGACCTCAGCTTCCCCGTGAAGTACTCAGCGCCGCGGCGGCCCACGGCCACCGGCGACTACGACAAGAAGAACTACGTCTGA.

In certain embodiments, the nucleobase polymer has a sequence of morethan 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides or nucleobasesor continuous nucleotide nucleobases that is the reverse complement ofthe protein encoding region of NM_001130861.1 or NM_003277.3, thenon-coding regions of NM_001130861.1 or NM_003277.3, SEQ ID NO: 2, orallelic variants thereof. In certain embodiments, the nucleobase polymeris less than 100, 50, or 25 nucleobases or base pairs. In certainembodiments, the nucleobase polymer is more than three nucleotides butless than seven or eight, or more than four nucleotides but less thanseven or eight, or more than five nucleotides but less than seven oreight.

In certain embodiments, the nucleobase polymer comprises monomers of(LNA) 1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol, ribose,deoxyribose, 2′-O-methy ribose, 2′-O-methoxyethyl ribose,2′-fluororibose, phosphodiester, phosphorothioate, methylphosphonate,phosphorodiamidate, piperazine phosphorodiamidate,P-(2-(hydroxymethyl)morpholino)-N,N-dimethylphosphonamidate,morpholin-2-ylmethanol, (2-(hydroxymethyl)morpholino)(piperazin-1-yl)phosphinate, or peptide nucleic acids or combinationsthereof.

In certain embodiments, pharmaceutical composition comprising apharmaceutically acceptable excipient and a nucleobase polymer disclosedherein. In certain embodiments, the nucleobase polymer comprises SEQ IDNO: 2 or fragment or reverse complement thereof. In certain embodiments,the nucleobase polymer is double or single stranded. In certainembodiments, the fragment is greater than 5, 10, 15, or 20 nucleotidesor nucleobases. In certain embodiments, the fragment is less than 100,50, or 25 nucleotides or nucleobases or base pairs.

In certain embodiments, the disclosure relates to synthetic,non-naturally occurring nucleobase polymer comprising a sequencedescribed herein or variants thereof. In certain embodiments, thevariant has 98%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 50% sequenceidentity thereto.

In certain embodiments, the disclosure relates to compositions, e.g.,pharmaceutical compositions, and uses reported herein comprising shorthairpin, synthetic, non-naturally occurring nucleobase polymercomprising TAGTTCTTCT (SEQ ID NO: 3), CGGTGGC(SEQ ID NO: 4), GGCGCTGA(SEQ ID NO: 5), GGAAGC(SEQ ID NO: 6), GACGGCCG (SEQ ID NO: 7),GCAGACC(SEQ ID NO: 8), ACAAGAGGCA (SEQ ID NO: 9), CCGCCCAGC(SEQ ID NO:10), CGCTGCG (SEQ ID NO: 11), ACACGGGCACA (SEQ ID NO: 12), ACGACAATG(SEQ ID NO: 13), AGCAGCCCGC(SEQ ID NO: 14), GAGGGCCACACG (SEQ ID NO:15), CGCCTTGGC(SEQ ID NO: 16), TGCACTGCGCG (SEQ ID NO: 17), AGGGTCACG(SEQ ID NO: 18), CACGGCG (SEQ ID NO: 19), GCCTGCACCTC(SEQ ID NO: 20),GAGTCGTACAC(SEQ ID NO: 21), ATGTGCCCGGT (SEQ ID NO: 22), CACAGCCCCTTCCA(SEQ ID NO: 23), GCGCCGTCACGA (SEQ ID NO: 24), TGCCACAT (SEQ ID NO: 25),ACGCCAGGATC(SEQ ID NO: 26), or combinations thereof optionally withmonomers of 1-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-7-ol and/orone or more phosphorothioate linkages.

In certain embodiments, the disclosure relates to a vector having anucleic acid encoding a hairpin comprising SEQ ID NO: 3-26. In certainembodiments, the disclosure contemplates methods of treating orpreventing respiratory distress comprising administering an effectiveamount of pharmaceutical agent comprising a vector having a nucleic acidencoding a hairpin RNA comprising SEQ ID NO: 3-26 to a subject in needthereof. In certain embodiments, the subject is diagnosed with alcoholiclung syndrome; acute respiratory distress syndrome; sepsis-associatedlung disorders; bacterial and viral pneumonia; ventilator induced lunginjury; bronchopulmonary dysplasia (BPD); asthma; bronchial, allergic,intrinsic, extrinsic or dust asthma; chronic or inveterate asthma; lateasthma or airways hyper-responsiveness; chronic obstructive pulmonarydisease (COPD); allergic rhinitis, bronchitis, or cystic fibrosis. Incertain embodiments, the peptide is administered in combination withanother respiratory agent, anti-inflammatory agent, or antibiotic.

Pharmaceutical Compositions

In certain embodiments, the disclosure contemplates pharmaceuticalcomposition comprising a peptide, or other pharmaceutical agentdisclosed herein, or pharmaceutically acceptable salts thereof, and apharmaceutically acceptable excipient. In certain embodiments, thepharmaceutical composition is in the form of a sterilized pH bufferedaqueous salt solution. In certain embodiments, the pharmaceuticallyacceptable excipient is aerosolizing agent or phospholipids. In certainembodiments, the aerosolizing agent is a hydrofluoroalkane,1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane, propane,n-butane, isobutene, carbon dioxide, air, nitrogen, nitrous oxide,dimethyl ether, trans-1,3,3,3-tetrafluoroprop-1-ene, or combinationsthereof. In certain embodiments, the phospholipid isdipalmitoylphosphatidylcholine, palmitoyl-oleoyl phosphatidylglycerol,phosphatidylglycerol, or combinations thereof.

In certain embodiments, the pharmaceutical compositions may be stored ina nebulizer, inhaler, or other container optionally sealed or under apressure for propelling the pharmaceutical agent(s). The container maycontain a spraying apparatus that is manually-actuated or pressurized.Metered dose inhalers (MDIs) typically have a handheld aerosol canisterthat, upon being pushed, releases an amount of medicine to inhale. Drypowder inhalers (DPIs) do not use a propellant to release the medicine.Instead, a dry powder form of the peptide or agent is drawn into yourlungs after a breath. In certain configurations, a container comprisingthe peptide or agent is inserted a device. Pressing a button or sectionon the device pierces the container. One can breathe in the powdercontained in the container through a mouthpiece on the device.

In certain embodiments, the pharmaceutical compositions may containnaturally or non-naturally occurring pulmonary surfactant compositions.Contemplated natural pulmonary surfactant compositions typicallycomprise 70-90% phospholipids (PC) such asdipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, andphosphatidylglycerol (PG); and 1-10% surfactant-associated proteins,apolipoproteins SP-A (SFTPA1), B (SFTPB), C (SFTPC) and D (SFTPD) (SPstanding for “surfactant-associated protein”); and 1-10% Cholesterol(neutral lipids). Artificial pulmonary surfactants include colfoscerilpalmitate (Exosurf), a mixture of DPPC with hexadecanol and tyloxapoladded as spreading agents; pumactant (Artificial Lung Expanding Compoundor ALEC), a mixture of DPPC and PG; KL-4, composed of DPPC,palmitoyl-oleoyl phosphatidylglycerol, and palmitic acid, combined witha 21 amino acid synthetic peptide that mimics the structuralcharacteristics of SP-B; and venticute, composed of DPPC, PG, palmiticacid and recombinant SP-C shares a nearly identical sequence with humanSP-C except that the palmitoylated cysteines are absent and have beenreplaced with phenylalanines to eliminate protein oligomerization.Contemplated animal derived surfactants include beractant (Alveofact),extracted from cow lung lavage fluid and (Survanta), extracted fromminced cow lung with additional DPPC, palmitic acid and tripalmitin;calfactant (Infasurf), extracted from calf lung lavage fluid; andporactant alfa (Curosurf)—extracted from material derived from mincedpig lung.

In certain embodiments, the pharmaceutical compositions disclosed hereinfurther comprise a respiratory agent selected from a glucocorticoidreceptor agonist (steroidal and non-steroidal) such as triamcinolone,triamcinolone acetonide, prednisone, mometasone furoate, loteprednoletabonate, fluticasone propionate, fluticasone furoate, fluocinoloneacetonide, dexamethasone cipecilate, desisobutyryl ciclesonide,clobetasol propionate, ciclesonide, butixocort propionate, budesonide,beclomethasone dipropionate, alclometasone dipropionate; a p38antagonist such as losmapimod; a phosphodiesterase (PDE) inhibitor suchas a methylxanthanine, theophylline, and aminophylline; a selective PDEisoenzyme inhibitor, a PDE4 inhibitor and the isoform PDE4D, such astetomilast, roflumilast, oglemilast, ibudilast, ronomilast; a modulatorof chemokine receptor function such as vicriviroc, maraviroc,cenicriviroc, navarixin; a leukotriene biosynthesis inhibitor,5-lipoxygenase (5-LO) inhibitor, and 5-lipoxygenase activating protein(FLAP) antagonist such as TA270(4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone) suchas setileuton, licofelone, quiflapon, zileuton, zafirlukast, ormontelukast; and a myeloperoxidase antagonist such as resveratrol andpiceatannol.

Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. For peptides or otheragents, the dosage administered to a patient is typically 0.0001 mg/kgto 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg,0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg,0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of thepatient's body weight. Further, the dosage and frequency ofadministration of peptides or agent may be reduced by enhancing uptakeand tissue penetration of the peptides or agents by modifications suchas, for example, lipidation and the inclusion of natural or artificialpulmonary surfactants.

The compositions include bulk drug compositions useful in themanufacture of pharmaceutical compositions (e.g., impure or non-sterilecompositions) and pharmaceutical compositions (i.e., compositions thatare suitable for administration to a subject or patient) which can beused in the preparation of unit dosage forms. Such compositions comprisea prophylactically or therapeutically effective amount of a prophylacticand/or therapeutic agent disclosed herein or a combination of thoseagents and a pharmaceutically acceptable carrier. In certainembodiments, the pharmaceutical compositions contain a pharmaceuticallyacceptable excipient that is a solubilizing agent such as a lipid,cholesterol, fatty acid, fatty acid alkyl ester, linoleic acid, oleicacid arachidonic acid, saccharide, polysaccharide, cyclodextrin,2-hydroxypropyl(cyclodextrin), or combinations thereof.

In certain embodiments, the pharmaceutically acceptable excipient isselected from lactose, sucrose, mannitol, triethyl citrate, dextrose,cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose,hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellosesodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose,povidone, methyl and ethyl acrylate copolymer, polyethylene glycol,fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin,glyceryl monooleate, silicon dioxide, titanium dioxide, talc, cornstarch, carnuba wax, stearic acid, sorbic acid, magnesium stearate,calcium stearate, castor oil, mineral oil, calcium phosphate, starch,carboxymethyl ether of starch, iron oxide, triacetin, acacia gum,esters, or salts thereof.

In certain embodiments, the pharmaceutical compositions is in solid formsurrounded by an enteric coating, i.e., a polymer barrier applied onoral medication that prevents its dissolution or disintegration in thegastric environment. Compounds typically found in enteric coatingsinclude methyl acrylate-methacrylic acid copolymers, cellulose acetatephthalate (CAP), cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methyl cellulose acetate succinate(hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP),methyl methacrylate-methacrylic acid copolymers, and combinationsthereof.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be bothnatural and artificial pulmonary surfactants, sterile liquids, such aswater and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions can be formulated as neutral or salt forms.Pharmaceutically acceptable salts include, but are not limited to, thoseformed with anions such as those derived from hydrochloric, phosphoric,acetic, oxalic, tartaric acids, etc., and those formed with cations suchas those derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

One embodiment provides a pharmaceutical pack or kit comprising one ormore containers filled with peptides or agents disclosed herein.Additionally, one or more other prophylactic or therapeutic agentsuseful for the treatment of a disease can also be included in thepharmaceutical pack or kit. One embodiment provides a pharmaceuticalpack or kit including one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

In certain embodiment, this disclosure contemplates pharmaceuticalcompositions comprising peptides and agents disclosed herein andpharmaceutically acceptable excipient. In certain embodiments, thisdisclosure contemplates the production of a medicament comprisingpeptides or agents disclosed herein and uses for methods disclosedherein.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising peptides and agents disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thecomposition is a pill or in a capsule or the composition is an aqueousbuffer, e.g., a pH between 6 and 8. In certain embodiments, thepharmaceutically acceptable excipient is selected from a filler,glidant, binder, disintegrant, lubricant, and saccharide.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents solventsor vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable (such as olive oil, sesame oil and viscoleo), preparationsincorporated into pulmonary surfactants (both natural and artificial),and injectable organic esters such as ethyl oleate.

Prevention of the action of microorganisms may be controlled by additionof any of various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. It may alsobe desirable to include isotonic agents, for example sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the peptides oragents may be admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or: (a) fillersor extenders, as for example, starches, lactose, sucrose, glucose,mannitol and silicic acid, (b) binders, as for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the peptides and agents, the liquid dosage forms may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acid esters ofsorbitan or mixtures of these substances, and the like.

In certain embodiments, production processes are contemplated which twocomponents, peptides and agents disclosed herein and a pharmaceuticalcarrier, are provided already in a combined dry form ready to bereconstituted together. In other embodiments, it is contemplated thatpeptides and agents disclosed herein and a pharmaceutical carrier areadmixed to provide a pharmaceutical composition.

Providing a pharmaceutic composition is possible in a one-step process,simply by adding a suitable pharmaceutically acceptable diluent to thecomposition in a container. In certain embodiments, the container ispreferably a syringe for administering the reconstituted pharmaceuticalcomposition after contact with the diluent. In certain embodiments, thecoated peptides or agents can be filled into a syringe, and the syringecan then be closed with the stopper. A diluent is used in an amount toachieve the desired end-concentration. The pharmaceutical compositionmay contain other useful component, such as ions, buffers, excipients,stabilizers, etc.

A “dry” pharmaceutical composition typically has only a residual contentof moisture, which may approximately correspond to the moisture contentof comparable commercial products, for example, has about 12% moistureas a dry product. Usually, the dry pharmaceutical composition accordingto the present invention has a residual moisture content preferablybelow 10% moisture, more preferred below 5% moisture, especially below1% moisture. The pharmaceutical composition can also have lower moisturecontent, e.g. 0.1% or even below. In certain embodiments, thepharmaceutical composition is provided in dry in order to preventdegradation and enable storage stability.

A container can be any container suitable for housing (and storing)pharmaceutically compositions such as inhalers, syringes, vials, tubes,etc. The pharmaceutical composition may then be applied via actuation orspecific needles of the syringe or via suitable catheters. A typicaldiluent comprises water for injection, and NaCl (preferably 50 to 150mM, especially 110 mM), CaCl₂ (preferably 10 to 80 mM, especially 40mM), sodium acetate (preferably 0 to 50 mM, especially 20 mM) andmannitol (preferably up to 10% w/w, especially 2% w/w). Preferably, thediluent can also include a buffer or buffer system so as to buffer thepH of the reconstituted dry composition, preferably at a pH of 6.2 to7.5, especially at pH of 6.9 to 7.1.

In certain embodiments, this disclosure contemplates a kit comprising apharmaceutical composition disclosed herein such as a peptide or agentand a container optionally with a suitable diluent. Further componentsof the kit may be instructions for use, administration means, such asinhalers, syringes, catheters, brushes, etc. (if the compositions arenot already provided in the administration means) or other componentsnecessary for use in medical (surgical) practice, such as substituteneedles or catheters, extra vials or further wound cover means. Incertain embodiments, the kit comprises a syringe housing the dry andstable hemostatic composition and a syringe containing the diluent (orprovided to take up the diluent from another diluent container).

In certain embodiments, the diluent is provided in a separate container.This can preferably be a syringe. The diluent in the syringe can theneasily be applied to the container for reconstitution of the drycompositions. If the container is also a syringe, both syringes can befinished together in a pack. It is therefore preferred to provide thedry compositions in a syringe, which is finished with a diluent syringewith a pharmaceutically acceptable diluent for reconstituting, said dryand stable composition.

Therapeutic Uses

This disclosure relates to compositions for use in managing respiratorydistress and related disorders. In certain embodiments, the disclosurerelates to methods of treating or preventing respiratory distresscomprising administering an effective amount of a pharmaceuticalcomposition comprising an active pharmaceutical agent disclosed hereinto a subject in need thereof. In certain embodiments, the disclosurecontemplates the use of pharmaceutical agents that prevent a claudin tocause claudin-18 to dissociate from ZO-1. In certain embodiments, thepharmaceutical agents decrease the concentration of claudin-5 in cellsand tissues of the lungs.

In one embodiment, compounds, compositions and methods of managing bytreatment or prevention of respiratory disorders comprisingadministering peptide or agents as described herein to a subject in needthereof. Respiratory disorders that may be prevented or treated includea disease or disorder of the respiratory system that can affect any partof the respiratory tract. Respiratory disorders include, but are notlimited to, a cold virus, bronchitis, pneumonia, tuberculosis,irritation of the lung tissue, hay fever and other respiratoryallergies, asthma, bronchitis, simple and mucopurulent chronicbronchitis, unspecified chronic bronchitis (including chronic bronchitisNOS, chronic tracheitis and chronic tracheobronchitis), emphysema, otherchronic obstructive pulmonary disease, asthma, status asthmaticus andbronchiectasis. Other respiratory disorders include allergic andnon-allergic rhinitis as well as non-malignant proliferative and/orinflammatory disease of the airway passages and lungs. Non-malignantproliferative and/or inflammatory diseases of the airway passages orlungs means one or more of (1) alveolitis, such as extrinsic allergicalveolitis, and drug toxicity such as caused by, e.g. cytotoxic and/oralkylating agents; (2) vasculitis such as Wegener's granulomatosis,allergic granulomatosis, pulmonary hemangiomatosis and idiopathicpulmonary fibrosis, chronic eosinophilic pneumonia, eosinophilicgranuloma and sarcoidoses.

In certain embodiments, the disclosure contemplates methods of treatingor preventing respiratory distress comprising administering an effectiveamount of a pharmaceutical composition comprising a pharmaceutical agentdisclosed herein to a subject in need thereof. In certain embodiments,the subject is diagnosed with alcoholic lung syndrome; acute respiratorydistress syndrome; sepsis-associated lung disorders; bacterial and viralpneumonia; ventilator induced lung injury; bronchopulmonary dysplasia(BPD); asthma; bronchial, allergic, intrinsic, extrinsic or dust asthma;chronic or inveterate asthma; late asthma or airwayshyper-responsiveness; chronic obstructive pulmonary disease (COPD);bronchitis; emphysema; allergic rhinitis; or cystic fibrosis. In certainembodiments, the peptide or agent disclosed herein is administered incombination with another respiratory agent.

In certain embodiments, the subject is at risk of, exhibit symptoms of,or diagnosed with a respiratory distress, disease or condition.

In certain embodiments, the subject is an infant. In certainembodiments, the subject is suffering BPD. In certain embodiments, thesubject is a premature infant. In certain embodiments, the subject isundergoing oxygen therapy as a protective agent.

In certain embodiments, the disclosure contemplates methods of treatingor preventing BPD comprising administering comprising administering aneffective amount of a pharmaceutical composition comprising a peptide oragent disclosed herein to a subject in need thereof. In certainembodiments, the subject is an infant suffering BPD or premature infantsundergoing oxygen therapy as a protective agent.

In certain embodiments, the respiratory agent selected from aglucocorticoid receptor agonist (steroidal and non-steroidal) such astriamcinolone, triamcinolone acetonide, prednisone, mometasone furoate,loteprednol etabonate, fluticasone propionate, fluticasone furoate,fluocinolone acetonide, dexamethasone cipecilate, desisobutyrylciclesonide, clobetasol propionate, ciclesonide, butixocort propionate,budesonide, beclomethasone dipropionate, alclometasone dipropionate; ap38 antagonist such as losmapimod; a phosphodiesterase (PDE) inhibitorsuch as a methylxanthanine, theophylline, and aminophylline; a selectivePDE isoenzyme inhibitor, a PDE4 inhibitor and the isoform PDE4D, such astetomilast, roflumilast, oglemilast, ibudilast, ronomilast; a modulatorof chemokine receptor function such as vicriviroc, maraviroc,cenicriviroc, navarixin; a leukotriene biosynthesis inhibitor,5-lipoxygenase (5-LO) inhibitor, and 5-lipoxygenase activating protein(FLAP) antagonist such as TA270(4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone) suchas setileuton, licofelone, quiflapon, zileuton, zafirlukast, ormontelukast; and a myeloperoxidase antagonist such as resveratrol andpiceatannol.

Methods of administering peptides and agents disclosed herein include,but are not limited to, pulmonary administration, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. See,e.g., U.S. Pat. Nos. 6,019,968; 5,985,200; 5,985,309; 5,934,272;5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos.WO 92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903. Ina specific embodiment, it may be desirable to administer thepharmaceutical compositions locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion, by injection, or by means of an implant, said implantbeing of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers. In certainembodiments, the aerosolizing agent or propellant is ahydrofluoroalkane, 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3-heptafluoropropane, propane, n-butane, isobutene, carbondioxide, air, nitrogen, nitrous oxide, dimethyl ether,trans-1,3,3,3-tetrafluoroprop-1-ene, or combinations thereof. In certainembodiments, the disclosure contemplates oral administration.

Methods of administering peptides and agents disclosed herein include,but are not limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epidural,and mucosal (e.g., intranasal and oral routes). In a specificembodiment, the peptides or agents are administered intramuscularly,intravenously, or subcutaneously. The compositions may be administeredby any convenient route, for example, by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local.

The peptides or agents of this disclosure may be administered incombination with other pharmaceutical agents such as antibiotics,anti-viral agents, anti-inflammatory agents, bronchodilators, ormucus-thinning medicines.

In certain embodiments, a bronchodilator for use as an additionaltherapeutic agent may be a short-acting beta2 agonist, a long-actingbeta2 agonist or an anticholinergic. In some embodiments, thebronchodilator is any one of, or combination of, salbutamol/albuterol,levosalbutamol/levalbuterol, pirbuterol, epinephrine, ephedrine,terbutaline, salmeterol, clenbuterol, formoterol, bambuterol,indacaterol, theophylline, tiotropium, or ipratropium bromide.

In certain embodiments, an antibiotic for use as an additionaltherapeutic agent may be any antibiotic chosen by a physician forreducing lung infections in a subject. In some embodiments, theantibiotic is any one of, or combination of, xicillin, clavulanatepotassium, aztreonam, ceftazidime, ciprofloxacin, gentamicin, ortobramycin.

EXPERIMENTS

Certain experiments reported herein are also reported in Schlingmann etal. Nat Commun. 2016, 7:12276. Recitation of this reference is not anadmission of prior art.

Chronic Alcohol Alters Lung Tight-Junction Permeability

The difference between AECs isolated from control- and alcohol-fedanimals (alcohol-exposed AECs) was evaluated using two differentmeasures of barrier function: transepithelial resistance (TER) andparacellular flux to soluble tracer molecules. Consistent with anincrease in paracellular leak, alcohol-exposed AECs had significantlydecreased TER and showed increased flux of both calcein (0.62 kDa) andTexas Red Dextran (10 kDa). Thus, alcohol exposure has a deleteriouseffect on AEC tight junctions.

As claudins are central to the regulation of tight-junctionpermeability, claudin protein composition of control- andalcohol-exposed AECs cultured on Transwell-permeable supports wasexamined by immunoblotting. The decrease in AEC barrier function inducedby alcohol correlated with decreased claudin-4 protein (FIG. 1A).Claudin-1, claudin-3 and claudin-7 were unaffected. However,AEC-associated claudins did not simply decrease in response to alcohol.Instead, claudin-5 was significantly increased in alcohol-exposed AECsas compared with control AECs (FIG. 1A). There also was a trend towardsincreased claudin-18 in alcohol-exposed AECs as compared with controlAECs (P=0.15, n=3, unpaired two-tailed t-test). As there was increasedparacellular leak accompanying increased claudin-5 expression, weexamined the effects of claudin remodelling in response to alcohol, todetermine whether this had a destabilizing effect on tight junctions.

Increased Claudin-5 Causes Increased Paracellular Leak

To confirm whether increased claudin-5 was sufficient to increaseparacellular leak, the dose response of increased yellow fluorescentprotein (YFP)-claudin-5 expression was examined using an adenovector totransduce primary AECs. A fourfold increase in claudin-5 expression((YFP−claudin-5+claudin-5)/claudin-5) significantly decreased TER (FIGS.1B and C) and increased paracellular flux. This level of YFP-claudin-5expression is in the physiologic range, comparable to the increase inendogenous AEC claudin-5 expression induced by alcohol (FIG. 1A). In theconverse experiment, lentiviral short hairpin RNA (shRNA) constructswere used to decrease claudin-5 expression.

Cldn-5 shRNA1 (Rat) 616-624 Sense (SEQ ID NO: 27) 5′ (NheI)CCCCCCAACGGCGATTACGACAATTCAAGAGATTGTCGTAATCGCCGTTG GTTTTTGG (PacI) 3′Anti (SEQ ID NO: 28) 5′ (PacI)CCAAAAACCAACGGCGATTACGACAATCTCTTGAATTGTCGTAATCGCCG TTGGGGGG (NheI) 3′Cldn-5 shRNA2 1594-1612 sense (SEQ ID NO: 29) 5′ (NheI)CCCCCCACCAAACTGCCGCTAACTTCAAGAGAGTTAGCGGCAGTTTGGTG GTTTTTGG (PacI) 3′anti (SEQ ID NO: 30) 5′ (PacI)CCAAAAACCACCAAACTGCCGCTAACTCTCTTGAAGTTAGCGGCAGTTTG GTGGGGGG (NheI) 3′scrambled sense (SEQ ID NO: 31) 5′ (NheI)CCCCAGTCATTGACGACAGCGTATTCAAGAGATACGCTGTCGTCAATGAC TTTTTTGG (PacI) 3′anti (SEQ ID NO: 32) 5′ (PacI)CCAAAAAAGTCATTGACGACAGCGTATCTCTTGAATACGCTGTCGTCAAT GACTGGGG (NheI) 3′

As shown in FIGS. 1D and E, using shRNA to decrease claudin-5 expressionby AECs from alcohol-fed rats caused a significant increase in TER andalso decreased paracellular flux.

As claudin-4 decreased in response to dietary alcohol, it could alsohave a negative impact on AEC barrier function in combination withincreased claudin-5. Thus, whether increased claudin-4 could rescue theeffects of alcohol on AECs was examined. Alcohol-exposed AECs transducedwith CFP-claudin-4 had only a partial increase in TER compared withcontrol AECs. Moreover, the effects of increased claudin-4 wereantagonized by a concurrent transduction with YFP-claudin-5. The factthat claudin-5 countered the ability of claudin-4 to promoteparacellular barrier function suggests that these claudins are directlyinteracting. Formation of complexes containing native claudin-4 andnative claudin-5 was confirmed by co-immunopurification analysis ofAECs. Using co-immunopurification, it was observed that native claudin-5directly interacts with native claudin-18 and ZO-1 indicating increasedclaudin-5 has a deleterious and dominant effect on other claudins andthereby impairs AEC barrier function.

Tight-Junction Spikes are Associated with Barrier Disruption

As revealed by immunofluorescence microscopy of claudin-18, AECs fromalcohol-fed rats have changes in tight-junction morphology, most notablyincreased formation of tight-junction spikes (FIG. 2A), which areactin-associated structures perpendicular to the axis of the cell-cellinterface that correlate with an increase in paracellular leak NormalAECs transduced to express increased claudin-5 also showed an increasein claudin-18 containing spikes, comparable to the effect of alcohol ontight-junction morphology (FIG. 2B). Morphologic disruption of tightjunctions was not restricted to claudin-18, as claudin-5 and ZO-1 werealso impaired in YFP-claudin-5-transduced AECs. To determine whetherZO-1 disruption was specifically linked to increased claudin-5, theeffect of increased YFP-claudin-3 on ZO-1 localization by AECs wasexamined. It was found that there was little effect on tight-junctionmorphology based on localization of claudin-18 or ZO-1. In acomplementary experiment, it was determined whether the ability ofalcohol to induce formation of tight junction spikes was antagonized bydepleting claudin-5 using shRNA. As shown in FIG. 2C, this was the casefor two different specific claudin-5 shRNAs. Thus, claudin-5 wassufficient to enhance formation of tight-junction spikes.

Although tight-junction spikes correlated with diminished paracellularbarrier function, how spikes were mechanistically linked to paracellularleak was not known. It is hypothesized that spikes represented areas ofenhanced tight-junction protein reorganization, which is known toincrease paracellular leak. To address this, AECs expressingYFP-claudin-18 that were adjacent to untransfected AECs were used. It isnoteworthy that YFP-claudin-18 acts to label tight-junction spikes inlive cells and did not induce formation of spikes in a manner comparableto claudin-5. Spike-associated YFP-claudin-18 was internalized byneighboring non-transduced cells, suggesting that the adjacent cellsinternalized claudin-18 from neighboring cells. Moreover,co-localization of ZO-1 to YFP-claudin-18 was variable, as there werereadily visualized YFP-claudin-18 structures that lacked co-localizationwith ZO-1, although claudin-18 and ZO-1 did co-localize in otherspike-associated structures.

To further characterize the behavior of claudins associated withtight-junction spikes, live-cell imaging microscopy of alcohol-exposedAECs transduced to express either YFP-claudin-5 or YFP-claudin-18 wasused, which revealed the dynamic nature of tight-junction spikes.Specifically, claudin-labelled vesicles were found to both fuse with andbud from tight-junction spikes. To further confirm that spikes weresites of active claudin vesicle formation and fusion, the effects of thedynamin inhibitor Dynasore 14 was examined on spike formation byalcohol-exposed AECs. Consistent with this, treatment with Dynasore at160 μM for 4 h caused a significant decrease in the number of cells withtight-junction spikes (FIG. 3A) comparable to the number of cellscontaining spikes observed for untreated control AECs (FIG. 3B).Dynasore-treated cells also showed an increase in punctateYFP-claudin-18 labelling, which probably represents secretory andendocytic vesicles that are inhibited from fusing with targetintracellular membranes by Dynasore. As an increase in tight-junctionspikes correlated with decreased barrier function, these data suggestthat increased vesicle-mediated trafficking of claudins both into andout of tight junctions contributes to paracellular leak in response toalcohol.

Claudin-5 Alters Interactions Between Claudin-18 and ZO-1

As tight junctions are multi-protein complexes, paracellular barrierfunction requires coordinating heterologous interactions betweentight-junction proteins. In intact cell junctions, protein—proteininteractions are reflected by co-localization of two or more proteins inthe same intracellular location when resolved at sufficient resolution.To understand how alcohol-induced changes affect tight junctions at amolecular level, AECs isolated from control- and alcohol-fed rats wereexamined by a form of super-resolution immunofluorescence microscopy,stochastic optical reconstruction microscopy (STORM), which has an X-Yresolution down to 20 nm. By the nature of the technique, STORM providesimages that are composed of point densities, resulting in a particulateimage at high magnification. STORM images obtained using the samelabelling and imaging conditions appeared to have differences in thesize of particulate clusters when comparing control versusalcohol-exposed AECs. Thus, the distribution of particulate clusterswere quantified. STORM imaging of normal AECs showed that claudin-18,claudin-5 and ZO-1 clusters had median areas of 1,240, 1,410 and 1,590nm², respectively. By contrast, alcohol-exposed AECs had claudin-18,claudin-5 and ZO-1 clusters with median areas of 1,410, 1,000 and 1,120nm², respectively. The alcohol-induced decrease in median cluster sizefor claudin-5 and ZO-1 was significant, as determined by Mann-WhitneyU-test; however, Claudin-18 cluster size was statistically unchanged. Asthese images were obtained using the same labelling and imagingconditions, the change in claudin-5 and ZO-1 cluster size induced byalcohol is likely to reflect tight-junction re-organization in responseto alcohol, despite the inability to assign a specific physiologiccorrelate to particulate clusters.

STORM images of AEC tight junctions showed a predominant linearintercellular complex with some projections and limited meshworkarchitecture. Some images also showed tight-junction spikes. STORManalysis of AECs did not show an extensive meshwork, as tight junctionsbetween adjacent type-I AECs in situ were shown to have a fairly limitedarchitecture. Moreover, STORM images are obtained using the totalinternal reflection fluorescence mode of illumination and thus anyjunctional elements perpendicular to the narrow plane of focus would notbe revealed using this approach. Here the STORM-imaging conditions wereoptimized for co-localization analysis between tight-junction proteinsas opposed to maximizing imaging resolution.

STORM enabled quantitative differences in co-localization to bemeasured, as these measurements were performed where cross-talk betweenthe two different channels was minimized. In alcohol-exposed AECs, therewas a significant decrease in co-localization between claudin-18 andZO-1 as compared with control AECs (FIG. 4A). Conversely, there was anincrease in co-localization between claudin-18 and claudin-5 in AECsisolated from alcohol-fed rats as compared with controls (FIG. 4B). Thisreciprocal relationship supports the hypothesis that in response tointeracting with claudin-5, claudin-18 dissociates from ZO-1.

To further investigate the alcohol-induced changes in ZO-1:claudin-18co-localization, AECs were examined using the PLA, which has a resolvingpower of 30-40 nm. PLA analysis of claudin-18 and ZO-1 in control AECsgave a robust signal. By contrast, alcohol-exposed AECs had asignificantly diminished PLA signal (FIG. 5A). Conversely, claudin-18and claudin-5 had a PLA signal that was increased in alcohol-exposedAECs as compared with control AECs (FIG. 5B). ZO-1:claudin-5co-localization was comparable for control and alcohol-exposed AECs,although the PLA signals have a slightly different appearance, becausethe cluster size for both ZO-1 and claudin-5 is sensitive to alcohol.These results parallel our analysis of the effects of alcohol onclaudin-18, claudin-5 and ZO-1 co-localization by STORM. Thus, twoindependent approaches demonstrate that ZO-1:claudin-18 proximity wasdiminished by alcohol and correlated with an increase inclaudin-18:claudin-5 proximity.

To determine whether increased claudin-5 was sufficient to decreaseassociation of claudin-18 and ZO-1, AECs transduced with YFP-claudin-5was examined by STORM. As opposed to untransduced AECs, where theco-localization index between claudin-18 and ZO-1 was 30.5%, AECsexpressing YFP-claudin-5 had significantly decreased co-localizationbetween claudin-18 and ZO-1 that was comparable to alcohol-exposed AECs.The significant drop in co-localization between ZO-1 and claudin-18 isconsistent with a decrease in interaction between these two proteins,which may alter the assembly state of claudin-18.

In AECs, both claudin-18 and ZO-1 are highly resistant to Triton X-100,suggesting that ZO-1:claudin-18 complexes are tightly associated withthe cytoskeleton. Thus, the effects of increased claudin-5 on theextractability of claudin-18, claudin-5 and ZO-1 were examined by TritonX-100. Consistent with previous measurements, less than ˜35% ofclaudin-18 can be solubilized by Triton X-100 under conditions where theinsoluble fraction primarily reflects proteins incorporated into tightjunctions 12 (FIG. 6A). By contrast, the majority of cell-associatedclaudin-5 is extractable by Triton X-100 (FIG. 6B).

When AECs were transduced with YFP-claudin-5, the Triton X-100 solublepool of claudin-18 significantly increased from 35.2 to 42.1representing a 20% increase in claudin-18 solubility (FIG. 6A). However,ZO-1 solubility was unchanged by increased claudin-5. Instead, theincrease in claudin-18 solubility induced by YFP-claudin-5 expression(FIG. 6A) correlated with the decrease in co-localization betweenclaudin-18 and ZO-1 from ˜31% to ˜16% as measured by STORM. Thisdecrease in co-localization suggests that decreased ZO-1:claudin-18interactions induced by increased claudin-5 are sufficient todestabilize the tight-junctional pool of claudin-18.

A Claudin-5 Peptide Improves Alveolar Barrier Function

An acetylated D-amino acid peptide corresponding to the region of thesecond extracellular (E2) domain directly adjacent to the thirdtransmembrane (TM3) domain of claudin-5, Ac-EFYDP (SEQ ID NO:1)-NH₂, wasprepared. The E2/TM3 region is implicated in mediating cis-claudininteractions, based on the crystal structure of claudin-15, as well asfunctional studies of claudin-3:claudin-5 and homomeric claudin-5interactions. In addition, the corresponding region of claudin-18, NFWMS(SEQ ID NO: 33) is not conserved and this region is sufficientlydivergent from the corresponding DFYNP (SEQ ID NO: 34) sequence found inother major claudins found in the lung, including claudin-3, -4 and -7.Claudin-1 does have an EFYDP (SEQ ID NO: 1) motif; however, it ispresent at low levels in AECs.

As shown in FIGS. 7B, D, and F, overnight incubation of alcohol-exposedAECs with the Ac-EFYDP (SEQ ID NO: 1)-NH₂ peptide increased barrierfunction, as measured by an increase in TER and decrease in paracellularflux of calcein and Texas Red Dextran. By contrast, control AECs wereunaffected by the Ac-EFYDP (SEQ ID NO: 1)-NH₂ peptide (FIG. 7A, C, E). Acontrol peptide, Ac-LYQY (SEQ ID NO:35)-NH₂, had no effect on AECbarrier function in either control or alcohol-exposed cells. The abilityof Ac-EFYDP (SEQ ID NO: 1)-NH₂ to improve the barrier function ofalcohol-exposed AECs correlated with a decrease in tight-junction spikeformation (FIG. 7G) and a specific decrease in total claudin-5 content(FIG. 7I). Claudin-18 and ZO-1 were unaffected (FIG. 7I) as wasclaudin-1. These data provide an additional demonstration that anincrease in endogenous claudin-5 diminishes AEC barrier function inresponse to alcohol indicating that targeting claudin-5 is a therapeuticapproach to prevent alcoholic lung syndrome.

1. A peptide having SEQ ID NO: 1 (EFYDP), derivatives, prodrugs, orsalts thereof.
 2. The peptide of claim 1, wherein proline (P) is aD-isomer, aspartic acid (D) is a D-isomer, tyrosine (Y) is a D-isomer,phenylalanine (F) is a D-isomer, glutamic acid (E) is a D-isomer, orcombinations thereof.
 3. The peptide of claim 2, wherein all of theamino acids are D-isomers.
 4. A pharmaceutical composition comprising apeptide of claim 1, or pharmaceutically acceptable salts thereof, and apharmaceutically acceptable excipient.
 5. The pharmaceutical compositionof claim 4, wherein the pharmaceutically acceptable excipient isaerosolizing agent or a phospholipid.
 6. The pharmaceutical compositionof claim 5, wherein the aerosolizing agent is a hydrofluoroalkane,1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoropropane, propane,n-butane, isobutene, carbon dioxide, air, nitrogen, nitrous oxide,dimethyl ether, trans-1,3,3,3-tetrafluoroprop-1-ene, or combinationsthereof.
 7. The pharmaceutical composition of claim 5, wherein thephospholipid is dipalmitoylphosphatidylcholine, palmitoyl-oleoylphosphatidylglycerol, phosphatidylglycerol, or combinations thereof. 8.The pharmaceutical composition of claim 4, further comprising anotherrespiratory agent.
 9. A method of treating or preventing respiratorydistress comprising administering an effective amount of apharmaceutical composition of claim 4 to a subject in need thereof. 10.The method of claim 9, wherein the subject is diagnosed with acuterespiratory distress syndrome; sepsis-associated lung disorders;bacterial and viral pneumonia; ventilator induced lung injury;bronchopulmonary dysplasia (BPD); asthma; bronchial, allergic,intrinsic, extrinsic or dust asthma; chronic or inveterate asthma; lateasthma or airways hyper-responsiveness; chronic obstructive pulmonarydisease (COPD); allergic rhinitis; bronchitis; emphysema; or cysticfibrosis.
 11. The method of claim 9, wherein the peptide is administeredin combination with another respiratory agent, anti-inflammatory agent,or antibiotic.
 12. A method of treating or preventing respiratorydistress comprising administering an effective amount of apharmaceutical composition comprisingN′-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthohydrazide or saltsthereof to a subject in need thereof.
 13. The method of claim 12,wherein the subject is diagnosed with alcoholic lung syndrome.
 14. Themethod of claim 12, whereinN′-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthohydrazide isadministered in combination with another respiratory agent,anti-inflammatory agent, or antibiotic.
 15. A method of treating orpreventing respiratory distress comprising administering an effectiveamount of a pharmaceutical composition comprising a nucleobase polymerconfigured to bind to mRNA of claudin 5 to a subject in need thereof.16. The method of claim 15, wherein the nucleobase polymer has asequence of more than 7 or more nucleotides or nucleobases or continuousnucleotide nucleobases that is the reverse complement of SEQ ID NO: 2.17. The method of claim 16, wherein the subject is diagnosed withalcoholic lung syndrome.
 18. The method of claim 16, wherein thenucleobase polymer is administered in combination with anotherrespiratory agent, anti-inflammatory agent, or antibiotic.